RESUMO
The design and selection of peptides targeting cellular proteins is challenging and often yields candidates with undesired properties. Therefore we deployed a new selection system based on the twin-arginine translocase (TAT) pathway of Escherichia coli, named hitchhiker translocation (HiT) selection. A pool of α-helix encoding sequences was designed and selected for interference with the coiled coil domain (CC) of a melanoma-associated basic-helix-loop-helix-leucine-zipper (bHLHLZ) protein, the microphthalmia associated transcription factor (MITF). One predominant sequence (iM10) was enriched during selection and showed remarkable protease resistance, high solubility and thermal stability while maintaining its specificity. Furthermore, it exhibited nanomolar range affinity towards the target peptide. A mutation screen indicated that target-binding helices of increased homodimer stability and improved expression rates were preferred in the selection process. The crystal structure of the iM10/MITF-CC heterodimer (2.1Å) provided important structural insights and validated our design predictions. Importantly, iM10 did not only bind to the MITF coiled coil, but also to the markedly more stable HLHLZ domain of MITF. Characterizing the selected variants of the semi-rational library demonstrated the potential of the innovative bacterial selection approach.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Membrana Transportadoras/química , Fator de Transcrição Associado à Microftalmia/química , Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Sequência de Aminoácidos , Sequência de Bases , Cristalografia por Raios X , Endopeptidase K/metabolismo , Proteínas de Escherichia coli/genética , Zíper de Leucina , Proteínas de Membrana Transportadoras/genética , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Biblioteca de Peptídeos , Multimerização Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SolubilidadeRESUMO
The twin-arginine translocation (TAT) pathway of the bacterial cytoplasmic membrane mediates translocation only of proteins that accomplished a native-like conformation. We deploy this feature in modular selection systems for directed evolution, in which folding helpers as well as dimeric or oligomeric protein-protein interactions enable TAT-dependent translocation of the resistance marker TEM ß-lactamase (ßL). Specifically, we demonstrate and analyze selection of (i) enhancers for folding by direct TAT translocation selection of a target protein interposed between the TorA signal sequence and ßL, (ii) dimeric or oligomeric protein-protein interactions by hitchhiker translocation (HiT) selection of proteins fused to the TorA signal sequence and to the ßL, respectively and (iii) heterotrimeric protein-protein interactions by combining HiT with protein fragment complementation selection of proteins fused to two split ßL fragments and TorA, respectively. The lactamase fragments were additionally engineered for improved activity and stability. Applicability was benchmarked with interaction partners of known affinity and multimerization whereby cellular fitness correlated well with biophysical protein properties. Ultimately, the HiT selection was employed to identify peptides, which specifically bind to leukemia- and melanoma-relevant target proteins (MITF and ETO) by coiled-coil or tetra-helix-bundle formation with high affinity. The various versions of TAT selection led to inhibiting peptides (iPEPs) of disease-promoting interactions and enabled so far difficult to achieve selections.